Interpretive Summary: In this communication, we merged three separate proteins produced by Mycobacterium avium subsp paratuberculosis into a single, long protein called a polyprotein (termed 74F in this study). This was done using recombinant DNA techniques. The three proteins have been shown to elicit protective responses in animals against TB infection, and therefore, the hypothesis was that these proteins might also be protective against infection with Mycobacterium avium subsp paratuberculosis, the bacterium that causes Johne’s disease. We evaluated protection using mice and showed that mouse tissues did have fewer lesions and bacteria load when compared to control mice.

Technical Abstract:
Johne’s Disease (JD) is a chronic infectious disease of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Here, we report the cloning and expression of a 74kDa recombinant polyprotein (Map74F) and its protective efficacy against MAP infection in mice. Map74F was generated by the sequential linkage of the ORFs of the~17.6-kDa C-terminal fragment of Map3527 to the full-length ORF of Map1519, followed at the C terminus with ~14.6-kDa N-terminal portion of Map3527. Mice immunized with Map74F had a significant IgG1 response but not IgG2a. In immunized animals, the IgG1/IgG2a ratio increased until 4 wk after MAP challenge. The ratio decreased from 8 wk indicating a shift to a Th1 response. Antigen specific IFN-gamma response, CD3+ and CD4+ T cells increased significantly in immunized mice. Following challenge, MAP burden was significantly lower in liver, spleen and mesenteric lymph nodes of immunized animals compared to control animals indicating protection against MAP infection. This was further evident by the improved liver and spleen pathology of the immunized animals, which had fewer granulomas and lower numbers of acid-fast bacilli. Results of this study indicated that immunization of mice with Map74F protected mice against MAP infection.